Preparation And Evaluation Of Ginsenoside Rg3 And Doxorubicin-loaded Micelles

Doxorubicin hydrochloride（DOX·HCl）is a cytotoxic drug that can act on DNA and has high activity against a variety of tumors.Ginsenoside Rg-3（G-Rg3）is a tetracyclic triterpenoid saponins sterol compound,which mainly exists in Ginseng and has anti-tumor effect.The combination of G-Rg3 with DOX·HCl has synergistic anti-tumor effects and can further weaken the toxicity of DOX·HCl.DOX·HCl is easily soluble in water,which is not conducive to the preparation of micelles.In this paper,the p H-sensitive and slightly water-soluble cis-aconitic anhydride doxorubiocin（CAD）was synthesized,and the structure of CAD was verified by ¹H-NMR and infrared spectroscopy（IR）.Poly（2-ethyl-oxazoline）-cholesteryl methycarbonated（PEOZ-CHMC）nano-micelles were constructed to co-encapsulate CAD and G-Rg3.The high performance liquid chromatography（HPLC）method for determine CAD and G-Rg3 in vitro.The detection wavelengths of CAD and G-Rg3 are 490 nm and 203 nm,respectively The two drugs have a good linear relationship in the concentration range of 1-40μg/m L and 10-120μg/m L,separately The precision and recovery rate meet the requirements of the experiment,which provides a basis for the quality evaluation of subsequent preparations.Blank micelles（BM）,CAD micelles（CAD-M）,G-Rg3 micelles（G-Rg3-M）and co-encapsulated CAD and G-Rg3 micelles（CAD/G-Rg3-M）were prepared by thin film dispersion method.Taking the drug loading as the index,the optimum formulation and process of micelles are as follows:the mass ratio of carrier to drug is 15:1:1（w/w）,the ratio of CAD to G-Rg3 is 1:1（w/w）,the ultrasound time is 5 minutes.The encapsulation efficiency of CAD and G-Rg3 are（61.41±3.66）%and（65.07±2.31）%,respectively.The average particle size of CAD/G-Rg3-M is about 100 nm.Based on scanning electron microscopy（SEM）and transmission electron microscopy（TEM）,micelles are spherical or quasi-spherical.The hydrolysis curve of CAD showed that DOX HCl.was dissociated from CAD under p H6.4,while there was no release in p H 7.4.The release of G-Rg3 was p H dependent.At p H 7.4,the release of CAD and G-Rg3 from micelles was slow and sustained in vitro.In order to increase the stability,the micelles were prepared as freeze-dried preparetions.The most optimal freeze-drying protectant is sucrose,and the optimal ratio of sucrose to micelle carrier was 4:1（w/w）.The appearance of the freeze-dried micelle was good and smooth.Compared with micelles the preparation before freeze-drying,there was no significantly change in the drug retention rate and light transmittance（P>0.05）.The morphology of the freeze-dried micelles was intact determined by SEM.Human malignant glioma U87 cell line and human breast cancer MCF-7 cell line were taken as the cell model,MTT assay was used to investigate the inhibitory effect of the micelles on the cells.The results showed that compared with CAD-M+G-Rg3-M and CAD+G-Rg3,CAD/G-Rg3-M has the strongest inhibitory effect on two cell models（P<0.05）.The inhibitory effect of CAD/G-Rg3-M on cells was enhanced when p H value decreased,which reflects the p H-sensitivity of micelles.The IC₅₀ value of CAD/G-Rg3-M was the lowest,which was consistent with the result of the cell inhibition.The synergistic effect of combined administration was evaluated by combination index（CI）,and the results showed that compared with CAD+G-Rg3,CAD/G-Rg3-M has moderate synergistic effects on the U87 cells and high synergetic effects on MCF-7 cells,which proved that CAD and G-Rg3 coloaded micelles have synergistic effects on U87 and MCF-7 cells.